Saccharides from bacteria have been used for many years in vaccines. As saccharides are T-independent antigens, however, they are poorly immunogenic. Conjugation to a carrier can convert T-independent antigens into T-dependent antigens, thereby enhancing memory responses and allowing protective immunity to develop. The most effective saccharide vaccines are therefore based on glycoconjugates, and the prototype conjugate vaccine was against Haemophilus influenzae type b (‘Hib’) [e.g. see chapter 14 of ref. 64].
Gram-negative bacteria are surrounded by an outer membrane that contains lipopolysaccharide. Lipopolysaccharides are a diverse group of molecules that act as endotoxins and elicit strong immune responses in mammals. Each lipopolysaccharide comprises three parts: an O-antigen (also referred to as the O-specific polysaccharide or O-polysaccharide), a core domain, and a lipid A domain. Antibodies against the O-antigen from a particular Gram-negative bacterium may confer protection against infection by that bacterium. Vaccines have therefore been envisaged that contain O-antigens conjugated to carrier proteins. For example, O-antigen-based conjugate vaccines have been proposed for various Salmonellae (e.g. serovars Salmonella Typhimurium and Salmonella Paratyphi A of Salmonella enterica in ref. 1 and 2); Shigella species [ref. 3, 4, 5, 6, 7, 8 and 9]; and Escherichia coli [ref. 10 and 11]. In these vaccines, the O-antigen is linked to the core domain from the full-length lipopolysaccharide (i.e. the conjugated polysaccharide is a lipopolysaccharide without its lipid A domain). The polysaccharide is conjugated to the carrier via its core domain. This core domain may itself induce protective antibodies, and conjugate vaccines have therefore been envisaged that contain a core domain that is not linked to an O-antigen [ref. 12].
Various methods are known for the conjugation of core domain-containing polysaccharides to a carrier protein. Some methods involve random activation of the polysaccharide chain (e.g. with cyanogen bromide (CNBr) or 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP)) prior to conjugation via a linker (see, for example, ref. 1 and 2). Other methods are more selective, involving a specific residue on the chain (e.g. the 2-keto-3-deoxyoctanoic acid (KDO) terminus of the core domain). For example, methods in reference 13 involve reductive amination between the carbonyl group in the KDO terminus with adipic acid dihydrazide (ADH) linker. Reactions involving reductive amination are typically very slow, e.g. the 7-day step in ref. 13. Another selective method is described in ref. 9, this time involving coupling via the carboxyl group in KOO using 1-ethyl-1-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and ADH. In these non-selective and selective methods, subsequent reaction with the carrier typically takes place using EDAC, which activates carboxyl groups in the protein for reaction with the linker. This EDAC activation can result in activation of the carboxyl group in the KDO subunit, leading to unwanted side reactions. The EDAC activation can also result in cross-linking of the carrier protein, because activated carboxyl groups in the protein can react with primary amine groups in the protein instead of in the linker.
Accordingly, there remains a need for further and better ways of preparing conjugates, particularly of the core domain from the lipopolysaccharide of Gram-negative bacteria.